[go: up one dir, main page]

US7588894B2 - Use of ID4 for diagnosis and treatment of cancer - Google Patents

Use of ID4 for diagnosis and treatment of cancer Download PDF

Info

Publication number
US7588894B2
US7588894B2 US11/345,836 US34583606A US7588894B2 US 7588894 B2 US7588894 B2 US 7588894B2 US 34583606 A US34583606 A US 34583606A US 7588894 B2 US7588894 B2 US 7588894B2
Authority
US
United States
Prior art keywords
cancer
methylation
sample
gene
tumor
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
US11/345,836
Other languages
English (en)
Other versions
US20070020646A1 (en
Inventor
Dave S. B. Hoon
Naoyuki Umetani
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
John Wayne Cancer Institute
Original Assignee
John Wayne Cancer Institute
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority to US11/345,836 priority Critical patent/US7588894B2/en
Application filed by John Wayne Cancer Institute filed Critical John Wayne Cancer Institute
Assigned to JOHN WAYNE CANCER INSTITUTE reassignment JOHN WAYNE CANCER INSTITUTE ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: UMETANI, NAOYUKI, HOON, DAVID S.B.
Publication of US20070020646A1 publication Critical patent/US20070020646A1/en
Priority to US12/417,577 priority patent/US7888033B2/en
Priority to US12/537,509 priority patent/US20130345144A1/en
Application granted granted Critical
Publication of US7588894B2 publication Critical patent/US7588894B2/en
Priority to US12/978,286 priority patent/US20110098341A1/en
Priority to US14/703,823 priority patent/US20160122826A1/en
Assigned to NATIONAL INSTITUTES OF HEALTH (NIH), U.S. DEPT. OF HEALTH AND HUMAN SERVICES (DHHS), U.S. GOVERNMENT reassignment NATIONAL INSTITUTES OF HEALTH (NIH), U.S. DEPT. OF HEALTH AND HUMAN SERVICES (DHHS), U.S. GOVERNMENT CONFIRMATORY LICENSE (SEE DOCUMENT FOR DETAILS). Assignors: JOHN WAYNE CANCER INSTITUTE
Assigned to NATIONAL INSTITUTES OF HEALTH (NIH), U.S. DEPT. OF HEALTH AND HUMAN SERVICES (DHHS), U.S. GOVERNMENT reassignment NATIONAL INSTITUTES OF HEALTH (NIH), U.S. DEPT. OF HEALTH AND HUMAN SERVICES (DHHS), U.S. GOVERNMENT CONFIRMATORY LICENSE (SEE DOCUMENT FOR DETAILS). Assignors: JOHN WAYNE CANCER INSTITUTE
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2537/00—Reactions characterised by the reaction format or use of a specific feature
    • C12Q2537/10—Reactions characterised by the reaction format or use of a specific feature the purpose or use of
    • C12Q2537/164—Methylation detection other then bisulfite or methylation sensitive restriction endonucleases
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/118—Prognosis of disease development
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/154—Methylation markers

Definitions

  • the present invention relates to inhibitors of DNA binding proteins. More specifically, the invention relates to use of ID4 for diagnosis and treatment of cancer.
  • CRC colorectal cancer
  • tumorsuppressor genes such as APC, p53, and genes on chromosome 18q (DCC, SMAD2, and DPC4/SMAD4) are inactivated by mutations or by chromosomal deletions (1, 2).
  • Some CRCs have microsatellite instability caused by inactivation of mismatch repair genes such as hMSH2 or hMLH1 (3).
  • epigenetic inactivation by hypermethylation of promoter regions of various tumor suppressor genes such as p16, APC, VHL, and hMLH1 have been found in CRCs (4-10) and used as molecular markers of CRC (11, 12).
  • Methylation of cytosines in CpG islands in the promoter region affects promoter activity and can down-regulate gene transcription (5). Because the promoter hypermethylation of genes in cancer cells is as significant as deletions or mutations (13-15), hypermethylation of key regulatory genes can play a significant role in transformation and tumor progression. Progression of transformed cells requires regulatory gene inactivation that promotes growth, dedifferentiation, invasion, and/or metastasis.
  • ID4 gene has a relation with growth and differentiation of cells, as reported with oligodendrocytes (31). Recently, it was reported that ID4 promoter is hypermethylated in 30% of primary gastric cancers, and expression is down-regulated in most gastric cancer cell lines by hypermethylation of the promoter region (32). Despite the structural similarity, ID4 is known to have some differences from the other three known ID gene members. Unlike ID1, ID2, and ID3, the immunoreactivity of which is significantly elevated in CRCs compared with normal epithelium (24), ID4 has a more restricted expression pattern during murine and avian embryogenesis and is expressed at more advanced stages of differentiation in tissues (18-20).
  • ID4 expression is restricted to the ventral part where cells grow slower, whereas other ID members are expressed in the dorsal part of the stomach where cells proliferate faster (18).
  • Information about ID4 function, expression, and regulation of tumor progression is very limited, and there are no published studies of ID4 in CRC to date.
  • axillary lymph node metastases The presence of axillary lymph node metastases is the most significant prognostic factor for patients with breast cancer (Fisher et al., 1993; Fitzgibbons et al., 2000). For patients with early breast cancers, the ability to predict nodal metastasis could alleviate the need for axillary staging.
  • the rate of nodal metastasis in T1 ( ⁇ 2.0 cm) breast cancers is reportedly from 18% to 31% (Barth et al., 1997; Carter et al., 1989; Holland et al., 1996).
  • risk factors of lymph node metastasis for invasive breast cancer There are several known risk factors of lymph node metastasis for invasive breast cancer to date. For nodal metastasis of T1 breast cancer, lymphovascular invasion, tumor size, histologic grade, histologic type, and estrogen receptor (ER) status are the major risk factors of pathological findings (Brenin et al., 2001
  • ID1 (Lin et al., 2000; Singh et al., 2002), and ID1 expression is associated with poor clinical outcome of patients with node-negative breast cancer (Schoppmann et al., 2003).
  • ID2 is low in invasive breast cancer and correlates with noninvasive phenotype (Itahana et al., 2003).
  • ID3 or ID4 in breast cancer has not been well studied. In breast cancer, animal studies have demonstrated that ID4 regulates mammary epithelial cell growth and differentiation, and overexpression of this protein in rat mammary gland carcinomas correlates with proliferation, invasiveness, and tumor weight (Shan et al., 2003).
  • certain human primary breast cancers reportedly have low or no expression of ID4 protein by immunohistochemistry (IHC) (Welcsh et al., 2002), suggesting downregulation of the ID4 gene.
  • IHC immunohistochemistry
  • the invention features a method of determining whether a subject is suffering from or at risk for developing colorectal cancer.
  • the method comprises obtaining a biological sample from a subject and determining the methylation level of an ID4 gene promoter, the expression level of an ID4 gene, or a combination thereof in the sample.
  • the methylation level of the ID4 gene promoter in the sample higher than a control methylation level, the expression level of the ID4 gene in the sample lower than a control expression level, or a combination thereof indicates that the subject is likely to be suffering from or at risk for developing colorectal cancer.
  • the subject may be suffering from or at risk for developing primary or metastatic colorectal cancer.
  • the invention features a method of determining the histopathological grade of colorectal cancer.
  • the method comprises obtaining a biological sample (e.g., a benign or primary colorectal tumor specimen sample) from a subject suffering from or suspected of being suffering from early stage colorectal cancer, determining the methylation level of an ID4 gene promoter, the expression level of an ID4 gene, or a combination thereof in the sample, and correlating the methylation level of the ID4 gene promoter, the expression level of the ID4 gene, or a combination thereof in the sample to a histopathological grade of the colorectal cancer.
  • a biological sample e.g., a benign or primary colorectal tumor specimen sample
  • determining the methylation level of an ID4 gene promoter, the expression level of an ID4 gene, or a combination thereof in the sample and correlating the methylation level of the ID4 gene promoter, the expression level of the ID4 gene, or a combination thereof in the sample to a histopathological grade of
  • the invention also provides a method of predicting the outcome of cancer, comprising obtaining a biological sample from a subject suffering from cancer, determining the methylation level of an ID4 gene promoter, the expression level of an ID4 gene, or a combination thereof in the sample, and correlating the methylation level of the ID4 gene promoter, the expression level of the ID4 gene, or a combination thereof in the sample to an outcome of the cancer.
  • the subject may suffer from or be at risk for developing American Joint Committee on Cancer stage I, II, III, or IV cancer.
  • the sample may be a primary tumor sample.
  • the outcome may be the survival of the subject after a surgical resection, e.g., a curative surgical resection.
  • the cancer may be any type of cancer such as colorectal cancer, breast cancer, pancreatic cancer, or small bowel cancer.
  • the invention further provides a method of identifying a compound for treating colorectal cancer.
  • the method comprises providing a colorectal cancer cell that contains an ID4 gene promoter, expresses an ID4 gene, or a combination thereof, contacting the cell with a test compound, and determining the methylation level of the ID4 gene promoter, the expression level of the ID4 gene, or a combination thereof in the cell.
  • the methylation level of the ID4 gene promoter in the cell lower than a control methylation level, the expression level of the ID4 gene in the cell higher than a control expression level, or a combination thereof indicates that the test compound is a candidate for treating colorectal cancer.
  • Also within the invention is a method of treating colorectal cancer, comprising identifying a subject suffering from or at risk for developing colorectal cancer according to the method described above and administering to the subject an effective amount of a compound that decreases the methylation level of the ID4 gene promoter, increases the expression level of the ID4 gene, or a combination thereof in the subject.
  • the invention features a method of determining whether a subject having cancer is suffering from or at risk for developing metastasis (e.g., lymph node metastasis).
  • the method comprises obtaining a biological sample from a subject having cancer and determining the methylation level of an ID4 gene promoter in the sample.
  • the methylation level of the ID4 gene promoter in the sample higher than a control methylation level indicates that the subject is likely to be suffering from or at risk for developing metastasis.
  • the invention provides a method of identifying a compound for treating breast cancer.
  • the method comprises providing a breast cancer cell that contains an ID4 gene promoter, contacting the cell with a test compound, and determining the methylation level of the ID4 gene promoter in the cell.
  • the methylation level of the ID4 gene promoter in the cell lower than a control methylation level indicates that the test compound is a candidate for treating breast cancer.
  • the cell is an early stage primary tumor cell.
  • the invention provides a method of treating cancer.
  • the method comprises identifying a subject having cancer and suffering from or at risk for developing lymph node metastasis according to the method described above and administering to the subject an effective amount of a compound that decreases the methylation level of the ID4 gene promoter, increases the expression level of the ID4 gene, or a combination thereof in the subject.
  • the invention also provides a method of treating breast cancer.
  • the method comprises identifying a subject suffering from or at risk for developing breast cancer, wherein the methylation level of an ID4 gene promoter in the subject is higher than a control methylation value, and administering to the subject an effective amount of a compound that decreases the methylation level of the ID4 gene promoter, increases the expression level of the ID4 gene, or a combination thereof in the subject.
  • the methylation level of the ID4 gene promoter may be determined by methylation-specific PCR, bisulfite sequencing, or a combination thereof.
  • the expression level of the ID4 gene may be determined at the mRNA level, the protein level, or a combination thereof.
  • the subject may be a human.
  • FIG. 1 Structure of the promoter region of ID4 gene and the primer design for methylation-specific PCR and bisulfite sequencing. CpG sites in the annealing site of methylation-specific PCR primers are indicated with “*”. (TSS, transcription start site)
  • FIG. 2 A. Representative bisulfite sequencing (reverse direction) of CRC (colorectal cancer) established cell lines (SW480 and DLD1) and peripheral blood lymphocytes in relation to the minimal promoter region. CpG sites (*) were fully methylated in SW480, mostly methylated in DLD1, and not methylated in peripheral blood lymphocytes. These findings corresponded to the methylation-specific PCR results.
  • FIG. 3 Representative immunohistochemistry of primary CRCs and normal colonic epithelium with their respective methylation-specific PCR results.
  • the vertical axes of methylation-specific PCR results are the fluorescent intensity representing the amount of PCR amplicon.
  • A normal colonic epithelium.
  • Diffusely stained cytoplasm represents high concentration of ID4 protein.
  • B adenoma. All adenomas were unmethylated.
  • Lightly stained cytoplasm represents ID4 protein.
  • C primary CRC determined as unmethylated by methylation-specific PCR.
  • Lightly stained cytoplasm represents ID4 protein. This tumor was well to moderately differentiated carcinoma.
  • D and E primary CRCs determined as methylated by methylation-specific PCR. Cytoplasm is not stained.
  • D and E tumors were poorly differentiated carcinoma and mucinous carcinoma, respectively. (U, unmethylated; M, methylated)
  • FIG. 4 Kaplan-Meier analysis of overall survival for CRC patients whose primary tumors were assessed for methylation status of ID4 promoter region.
  • FIG. 6 Representative capillary array electrophoresis (CAE) results of methylation-specific PCR (MSP).
  • MSP methylation-specific PCR
  • the fluorescent intensity shown on the vertical axis represents the amount of PCR amplicon assessed by CAE.
  • the horizontal axis represents the fragment size of the amplicon.
  • ‘M’ peak methylated-specific product of MSP
  • ‘U’ peak unmethylated-specific product of MSP.
  • FIG. 7 ID4 transcription level and MI in cell lines. ID4 mRNA transcription level and MI in eight breast cell lines (•) and additional three colorectal cancer cell lines (o).
  • Vertical axis represents the relative ID4 mRNA level normalized by GAPDH mRNA level in log scale. Horizontal axis represents MI values.
  • FIG. 8 Matched background parameters in T1 breast cancers. Distribution of size and patient age, matched background parameters between N (+) group and N ( ⁇ ) group, with mean diamonds indicating group mean values and 95% confidence intervals of mean. Each dot represents the age and tumor size of each tumor.
  • FIG. 9 MI (methylation index) distribution in N (+) and N ( ⁇ ) breast cancers. Methylation status of each specimen was determined by MI calculated from the fluorescent signal intensities of MSP. MI was measured twice for each specimen; values larger than 0.1 corresponded to hypermethylation. Dotted horizontal line represents the threshold level of hypermethylation. Each dot represents the MI of each tumor.
  • ID4 gene is a member of the inhibitor of DNA binding (ID) family proteins that inhibit DNA binding of basic helix-loop-helix transcription factors.
  • ID4 gene is a member of the inhibitor of DNA binding (ID) family proteins that inhibit DNA binding of basic helix-loop-helix transcription factors.
  • ID4 gene was assessed for the epigenetic inactivation of ID4 gene on colorectal cancer (CRC) development and its clinical significance was assessed.
  • ID4 methylation status of the promoter region was assessed by methylation-specific PCR and bisulfite sequencing.
  • the mRNA expression level was assessed by quantitative real-time reverse transcription-PCR.
  • the methylation status of 9 normal epithelia, 13 adenomas (benign colorectal disease), 92 primary CRCs, and 26 liver metastases was assessed by methylation-specific PCR.
  • ID4 protein expression was assessed by immunohistochemistry analysis of tissue specimen.
  • CRC cell lines were shown to be hypermethylated, and mRNA expression was suppressed and could be restored by 5-aza-cytidine treatment.
  • the frequency of ID4 hypermethylation was 0 of 9 (0%), 0 of 13 (0%), 49 of 92 (53%), and 19 of 26 (73%), respectively, with a significant elevation according to CRC pathological progression.
  • Immunohistochemistry analysis showed ID4 expression of normal colon epithelia, adenomas, and unmethylated primary CRCs but not hypermethylated CRC specimens.
  • AJCC American Joint Committee on Cancer
  • ID4 gene is a potential tumor suppressor gene for which methylation status may play an important role in the CRC progression.
  • ID4 protein reportedly have low or no expression of ID4 protein, but its role in carcinogenesis and cancer progression is unknown.
  • MSP methylation-specific PCR
  • the invention provides various cancer diagnostic methods.
  • One method is used to determine whether a subject is suffering from or at risk for developing colorectal cancer.
  • a biological sample is obtained from a subject and the methylation level of an ID4 gene promoter, the expression level of an ID4 gene, or a combination thereof in the sample is determined. If the methylation level of the ID4 gene promoter in the sample is higher than a control methylation level, the expression level of the ID4 gene in the sample is lower than a control expression level, or a combination thereof, the subject is likely to be suffering from or at risk for developing colorectal cancer, e.g., primary or metastatic colorectal cancer.
  • the control methylation level of the ID4 gene promoter and the control expression level of the ID4 gene may be, for example, the methylation level and the expression level detected in a normal epithelia sample or an adenomas sample.
  • a biological sample e.g., a benign lesion sample, an early stage primary tumor sample, or a T1, T2, or T3 breast cancer sample
  • a subject having cancer e.g., breast cancer, pancreatic cancer, colorectal cancer, or gastric cancer.
  • the methylation level of an ID4 gene promoter in the sample is determined. If the methylation level of the ID4 gene promoter in the sample is higher than a control methylation level, the subject is likely to be suffering from or at risk for developing metastasis.
  • the expression level of the ID4 gene in the sample is lower than a control expression level.
  • the control methylation level of the ID4 gene promoter may be, for example, the methylation level detected in a normal tissue sample, a primary tumor sample obtained from a subject without metastasis, or an isolated standard ID4 DNA.
  • An early stage tumor refers to a small tumor that has not invaded other tissues, i.e., no metastasis has occurred.
  • the methylation level of a gene promoter can be determined, for example, by methylation-specific PCR, bisulfite sequencing (COBRA), or pyrosequencing.
  • Methylation-specific PCR is a technique whereby DNA is amplified by PCR dependent upon the methylation state of the DNA. Determining the methylation state of a nucleic acid includes amplifying the nucleic acid by means of oligonucleotide primers that distinguishes between methylated and unmethylated nucleic acids. MSP can rapidly assess the methylation status of virtually any group of CpG sites within a CpG island, independent of the use of methylation-sensitive restriction enzymes.
  • This assay entails initial modification of DNA by sodium bisulfite, converting all unmethylated, but not methylated, cytosines to uracil, and subsequent amplification with primers specific for methylated versus unmethylated DNA.
  • MSP requires only small quantities of DNA, is sensitive to 0.1% methylated alleles of a given CpG island locus, and can be performed on DNA extracted from paraffin-embedded samples. MSP eliminates the false positive results inherent to previous PCR-based approaches which relied on differential restriction enzyme cleavage to distinguish methylated from unmethylated DNA. This method is very simple and can be used on small amounts of tissue or few cells and fresh, frozen, or paraffin-embedded sections. MSP product can be detected by gel electrophoresis, capillary array electrophoresis, or real-time quantitative PCR.
  • Gene expression can be detected and quantified at mRNA or protein level using a number of means well known in the art.
  • cells in biological samples e.g., cultured cells, tissues, and body fluids
  • RNA levels in the lysates or in RNA purified or semi-purified from the lysates determined by any of a variety of methods familiar to those in the art.
  • Such methods include, without limitation, hybridization assays using detectably labeled gene-specific DNA or RNA probes and quantitative or semi-quantitative RT-PCR (e.g., real-time PCR) methodologies using appropriate gene-specific oligonucleotide primers.
  • quantitative or semi-quantitative in situ hybridization assays can be carried out using, for example, unlysed tissues or cell suspensions, and detectably (e.g., fluorescently or enzyme-) labeled DNA or RNA probes.
  • detectably e.g., fluorescently or enzyme-
  • Additional methods for quantifying mRNA levels include RNA protection assay (RPA), cDNA and oligonucleotide microarrays, and colorimetric probe based assays.
  • Some of these protein-measuring assays can be applied to bodily fluids or to lysates of test cells, and others (e.g., immunohistological methods or fluorescence flow cytometry) applied to unlysed tissues or cell suspensions. Methods of measuring the amount of a label depend on the nature of the label and are known in the art.
  • Appropriate labels include, without limitation, radionuclides (e.g., 125 I, 131 I, 35 S, 3 H, or 32 P), enzymes (e.g., alkaline phosphatase, horseradish peroxidase, luciferase, or ⁇ -glactosidase), fluorescent moieties or proteins (e.g., fluorescein, rhodamine, phycoerythrin, GFP, or BFP), or luminescent moieties (e.g., QdotTM nanoparticles supplied by the Quantum Dot Corporation, Palo Alto, Calif.).
  • Other applicable assays include quantitative immunoprecipitation or complement fixation assays.
  • the invention also provides various cancer prognostic methods.
  • One method is used to determine the histopathological grade of colorectal cancer.
  • a biological sample e.g., a benign or primary colorectal tumor specimen sample
  • the methylation level of an ID4 gene promoter, the expression level of an ID4 gene, or a combination thereof in the sample is determined and correlated to a histopathological grade of the colorectal cancer.
  • a biological sample e.g., a primary tumor sample
  • cancer e.g., colorectal cancer, breast cancer, pancreatic cancer, or small bowel cancer.
  • the methylation level of an ID4 gene promoter, the expression level of an ID4 gene, or a combination thereof in the sample is determined and correlated to an outcome of the cancer.
  • the subject is suffering from or at risk for developing American Joint Committee on Cancer (AJCC) stage I, II, III, or IV cancer.
  • AJCC American Joint Committee on Cancer
  • the outcome is the survival of the subject after a surgical resection, i.e., a noncurative or curative surgical resection.
  • the invention further provides methods for screening cancer treatment drugs.
  • the method is used to identify a compound for treating colorectal cancer.
  • the method involves the steps of providing a colorectal cancer cell that contains an ID4 gene promoter, expresses an ID4 gene, or a combination thereof, contacting the cell with a test compound, and determining the methylation level of the ID4 gene promoter, the expression level of the ID4 gene, or a combination thereof in the cell. If the methylation level of the ID4 gene promoter in the cell is lower than a control methylation level, the expression level of the ID4 gene in the cell is higher than a control expression level, or a combination thereof, the test compound is identified as a candidate for treating colorectal cancer.
  • the control methylation level and the control expression level may be, for example, the methylation level and the expression level detected in the colorectal cancer cell prior to the contacting step.
  • the method is used to identify a compound for treating breast cancer.
  • the method involves the steps of providing a breast cancer cell (e.g., an early stage primary tumor cell) that contains an ID4 gene promoter, contacting the cell with a test compound, and determining the methylation level of the ID4 gene promoter in the cell. If the methylation level of the ID4 gene promoter in the cell is lower than a control methylation level, the test compound is identified as a candidate for treating breast cancer.
  • the control methylation level may be, for example, the methylation level detected in the breast cancer cell prior to the contacting step.
  • test compounds of the present invention can be obtained using any of the numerous approaches (e.g., combinatorial library methods) known in the art. See, e.g., U.S. Pat. No. 6,462,187.
  • libraries include, without limitation, peptide libraries, peptoid libraries (libraries of molecules having the functionalities of peptides, but with a novel, non-peptide backbone that is resistant to enzymatic degradation), spatially addressable parallel solid phase or solution phase libraries, synthetic libraries obtained by deconvolution or affinity chromatography selection, and the “one-bead one-compound” libraries.
  • Compounds in the last three libraries can be peptides, non-peptide oligomers, or small molecules. Examples of methods for synthesizing molecular libraries can be found in the art. Libraries of compounds may be presented in solution, or on beads, chips, bacteria, spores, plasmids, or phages.
  • a cancer cell or a subject that contains an ID4 gene promoter, expresses an ID4 gene, or a combination thereof is provided.
  • the cell or subject may be a cell or subject that naturally contains an ID4 gene promoter, expresses an ID4 gene, or a combination thereof, or alternatively, a cell or subject that contains a recombinant form of an ID4 gene promoter, expresses a recombinant form of an ID4 gene, or a combination thereof.
  • compositions typically include the compounds and pharmaceutically acceptable carriers.
  • “Pharmaceutically acceptable carriers” include solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.
  • Other active compounds e.g., taxol, doxorubicin, or 5-FU
  • taxol doxorubicin, or 5-FU
  • a pharmaceutical composition is formulated to be compatible with its intended route of administration. See, e.g., U.S. Pat. No. 6,756,196.
  • routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration.
  • Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • the compounds are prepared with carriers that will protect the compounds against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • a controlled release formulation including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art.
  • the materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc.
  • Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the invention provides methods for treating cancer.
  • the method is used to treat colorectal cancer.
  • the method involves the steps of identifying a subject suffering from or at risk for developing colorectal cancer according to the method described above and administering to the subject an effective amount of a compound that decreases the methylation level of the ID4 gene promoter, increases the expression level of the ID4 gene, or a combination thereof in the subject.
  • the method is used to treat cancer.
  • the method involves the steps of identifying a subject having cancer and suffering from or at risk for developing lymph node metastasis according to the method described above and administering to the subject an effective amount of a compound that decreases the methylation level of the ID4 gene promoter, increases the expression level of the ID4 gene, or a combination thereof in the subject.
  • the method is used to treat breast cancer.
  • a subject suffering from or at risk for developing breast cancer is identified where the methylation level of an ID4 gene promoter in the subject is higher than a control methylation value.
  • An effective amount of a compound is administered to the subject to decrease the methylation level of the ID4 gene promoter, increase the expression level of the ID4 gene, or a combination thereof in the subject.
  • the control methylation level may be, for example, the methylation level detected in a normal tissue sample.
  • treating is defined as administration of a substance to a subject with the purpose to cure, alleviate, relieve, remedy, prevent, or ameliorate a disorder, symptoms of the disorder, a disease state secondary to the disorder, or predisposition toward the disorder.
  • a subject to be treated may be identified, e.g., using the diagnostic methods described above.
  • an “effective amount” is an amount of a compound that is capable of producing a medically desirable result in a treated subject.
  • the medically desirable result may be objective (i.e., measurable by some test or marker) or subjective (i.e., subject gives an indication of or feels an effect).
  • the treatment methods can be performed alone or in conjunction with other drugs and/or radiotherapy. See, e.g., U.S. Patent Application 20040224363.
  • a therapeutic compound e.g., a compound that decreases the methylation level of an ID4 gene promoter, increases the expression level of an ID4 gene, or a combination thereof in a subject
  • the therapeutic compound may be an ID4 protein or a nucleic acid encoding an ID4 protein.
  • the therapeutic compound can be a demethylating agent such as 5-aza-cytidine or a compound capable of demethylating CpG islands methylated in promoter regions. These compounds can reverse gene silencing and activate gene expression.
  • HDAC histone deacetylase
  • the compound will be suspended in a pharmaceutically-acceptable carrier and administered orally, or by intravenous (i.v.) infusion, or injected or implanted subcutaneously, intramuscularly, intrathecally, intraperitoneally, intrarectally, intravaginally, intranasally, intragastrically, intratracheally, or intrapulmonarily.
  • the compound is preferably delivered directly to tumor cells, e.g., to a tumor or a tumor bed following surgical excision of the tumor, in order to kill any remaining tumor cells.
  • the compound can be administered to, for example, a subject that has not yet developed detectable invasion and metastases but is found to have increased methylation level of an ID4 gene promoter, decreased expression level of an ID4 gene, or a combination thereof.
  • the dosage required depends on the choice of the route of administration, the nature of the formulation, the nature of the subject's illness, the subject's size, weight, surface area, age, and sex, other drugs being administered, and the judgment of the attending physician. Suitable dosages are in the range of 0.01-100.0 mg/kg. Wide variations in the needed dosage are to be expected in view of the variety of compounds available and the different efficiencies of various routes of administration.
  • oral administration would be expected to require higher dosages than administration by i.v. injection. Variations in these dosage levels can be adjusted using standard empirical routines for optimization as is well understood in the art.
  • Encapsulation of the compound in a suitable delivery vehicle e.g., polymeric microparticles or implantable devices may increase the efficiency of delivery, particularly for oral delivery.
  • polynucleotides are administered to a subject.
  • Polynucleotides can be delivered to target cells by, for example, the use of polymeric, biodegradable microparticle or microcapsule devices known in the art. Another way to achieve uptake of the nucleic acid is using liposomes, prepared by standard methods.
  • the polynucleotides can be incorporated alone into these delivery vehicles or co-incorporated with tissue-specific or tumor-specific antibodies.
  • naked DNA (i.e., without a delivery vehicle) can also be delivered to an intramuscular, intradermal, or subcutaneous site.
  • a preferred dosage for administration of polynucleotide is from approximately 10 6 to 10 12 copies of the polynucleotide molecule.
  • ID4 gene may be expressed in normal colon epithelium and have a putative tumor suppressive role in CRC, contrary to other ID members.
  • ID4 gene transcription is silenced during CRC development and that hypermethylation of the ID4 promoter region is one of the main mechanisms of inactivation.
  • Cells were seeded at 7 ⁇ 10 5 /T-25 flask on day zero; the culture medium was changed on day two, and cells were then treated with 5Aza at final concentrations of 5 ug/mL for 48 hours (SW480 and DLD1) and 10 ug/mL for 72 hours (DLD1). After treatment, cells were harvested for DNA and RNA as described above.
  • Mg 2+ concentration was 1.5 mmol/L for methylation-specific primer set and 2.5 mmol/L for unmethylation-specific primer set.
  • Primer concentration was 0.1 mmol/L for methylation-specific primer set and 0.4 mmol/L for unmethylation-specific primer set.
  • PCR products were detected and analyzed by CEQ 8000XL capillary array electrophoresis system (Beckman Coulter, Inc., Fullerton, Calif.) with CEQ 8000 software version 6.0 (Beckman Coulter) as described previously (35). Methylation status was determined by the ratio of the signal intensities of methylated and unmethylated PCR products; samples with methylated to unmethylated ratio larger than 0.2 were determined as methylated. Each primer set was confirmed not to yield amplification on DNA without bisulfite treatment.
  • PCR amplification was done in a 50-uL reaction volume with 2 uL template for 40 cycles of 30 seconds at 94° C., 30 seconds at 60° C., and 30 seconds at 72° C., followed by a 7-minute final extension at 72° C. with the use of 2.5 mmol/L of Mg 2+ .
  • Reverse-transcriptase reactions were done on 1.0 ug of extracted total RNA with Moloney murine leukemia virus reverse-transcriptase (Promega, Madison, Wis.) with oligodeoxythymidylic acid primers, as described previously (39). Quantitative real-time reverse transcription-PCR assay was done on the iCycler iQ Real-Time thermocycler detection system (Bio-Rad Laboratories, Hercules, Calif.; 39).
  • the reaction mixture consisted of cDNA template synthesized by reverse-transcription from 250 ng of total RNA, 0.2 mmol/L of forward primer (5′-CGCTCACTGCGCTCAACAC-3′), 0.2 mmol/L of reverse primer (5′-TCAGGCGGCCGCACACCT-3′), and 0.6 mmol/L of fluorescence resonance energy transfer probe (5′-FAM-CATTCTGTGCCGCTGAGCCG-BHQ-3′).
  • PCR amplification was done in a 20-ul reaction volume for 45 cycles of 30 seconds at 94° C., 30 seconds at 58° C., and 30 seconds at 72° C. with 3 mmol/L of Mg 2+ .
  • Absolute copy numbers were determined by a standard curve with serial dilutions (10 8 to 10 1 copies) of DNA containing ID4 or GAPDH cDNA sequence. Analysis without templates was done as a negative control in each study. PCR products were electrophoresed on 2% agarose gels to confirm correct product size and absence of nonspecific bands. The expression level of the housekeeping gene GAPDH was measured as an internal reference with a standard curve to determine the integrity of template RNA for all of the specimens. The ratio of ID4 and GAPDH mRNA level was calculated as follows: (absolute copy number of ID4)/(absolute copy number of GAPDH) as described previously (39).
  • Immunohistochemistry Immunohistochemistry analysis of ID4 protein expression in primary CRCs, adenomas, and normal colon tissue sections was done to determine concordance with methylation-specific PCR results. Immunohistochemistry was done on 3-um sections of formalin-fixed, paraffin-embedded tissues, which were placed on silanecoated slides and baked at 60° C. for 1 hour. Afterward, the slides were deparaffinized, hydrated, and placed in antigen retrieval buffer (DAKO Corporation, Carpinteria, Calif.) at 95° C. for 10 minutes. Endogenous peroxidase activity was quenched by 1% hydrogen peroxide for 10 minutes.
  • antigen retrieval buffer DAKO Corporation, Carpinteria, Calif.
  • ID4 mRNA expression was not detected.
  • the partially hypermethylated LOVO cell line had 1.4 ⁇ 10 5 copies/250 ng of total RNA, and the ID4 to GAPDH expression ratio was 2.5 ⁇ 10 ⁇ 2 (Table 1).
  • ID4 hypermethylation was not detected in nine normal colon epithelia or in 13 adenomas, but it was identified in 49 of 92 (53%) primary CRCs and in 19 of 26 (73%) liver metastases.
  • the frequency of hypermethylation was significantly increased as the tumor progressed from adenoma to primary carcinoma and then to metastatic CRC(P ⁇ 0.0001 by x 2 test; P ⁇ 0.0001 by Cochran-Armitage trend test; Table 2).
  • Immunohistochemistry Of the 10 primary CRCs analyzed with immunohistochemistry, six were hypermethylated and four were unmethylated. In FIG. 3 , representative immunohistochemistry results are aligned with corresponding methylation-specific PCR results. Normal colonic epithelia and adenomas had diffusely stained cytoplasm, representing high concentration of ID4 protein expression ( FIGS. 3 , A and B). In primary CRCs determined as unmethylated by methylation-specific PCR, cell cytoplasm was lightly stained for ID4 protein. A representative microscopic picture of an unmethylated primary CRC, which was well to moderately differentiated carcinoma, is shown in FIG. 3C .
  • cytoplasm of primary CRCs determined as hypermethylated by methylation-specific PCR did not immunostain.
  • Representative microscopic pictures of hypermethylated primary CRCs, which were poorly differentiated carcinoma and mucinous carcinoma, are shown in FIG. 3 , D and E, respectively. No nuclear staining was observed in any type of tissues.
  • Methylation status of primary CRCs was independent of sex, age, tumor location, tumor diameter, American Joint Committee on Cancer tumor-node-metastasis (TNM) scores, and stage (Table 3).
  • TNM tumor-node-metastasis
  • methylation status, TNM N and methylated score, and tumor diameter were selected as covariates that had P values under 0.1 in univariate analysis.
  • TNM stage was not selected because of the direct association with the TNM N and methylated score, and histopathological tumor grade was not selected because it was highly dependent on ID4 methylation status.
  • ID4 is suggested as one of the key controlling factors for cell differentiation, we hypothesized that epigenetic regulation of ID4 gene might affect tumor differentiation and progression of CRC. The relationship between ID4 promoter hypermethylation and mRNA transcription, protein expression levels, and clinicopathological characteristics was determined.
  • ID4 protein expression level in CRCs we did immunohistochemistry study on a subset of specimens that were methylated or unmethylated by methylation-specific PCR. ID4 protein was diffusely expressed in cytoplasm of nonmalignant epithelium, adenomas, and unmethylated CRCs, but it tended to be unexpressed in hypermethylated CRCs. Concordance between immunohistochemistry and methylation-specific PCR results showed that aberrant hypermethylation of ID4 down-regulated protein expression.
  • Methylation analysis on tissue specimens was done in a blinded fashion without any clinical information.
  • the methylation status was determined by methylation-specific PCR based on the intensity ratio of the methylation-specific and unmethylation-specific peaks by automated capillary array electrophoresis system with analysis software. Because methylation-specific PCR can detect a very small percentage of methylated DNA in abundant unmethylated DNA, the methylation-specific PCR results were methylation-positive, even if only a small part of the microdissected tumor cells was hypermethylated. Aberrant hypermethylation was not found in normal colonic epithelia and adenomas, whereas 53% of primary CRCs and 73% of liver metastases were hypermethylated.
  • ID4 gene could be a putative tumor suppressor gene of CRC, which is epigenetically inactivated by promoter hypermethylation at a later stage of cancer development.
  • the absence of hypermethylation in adenomas, which are considered to be precursors of CRC (1, 40), and the high frequency of hypermethylation in primary CRCs support the concept that ID4 downregulation is related to the malignant transformation of neoplastic tumor cells.
  • Multivariate analysis by Cox's proportional hazard model identified hypermethylation of ID4 as a significant independent risk factor of poor prognosis after curative surgical resection.
  • ID4 methylation status had higher impact on prognosis than lymph node metastasis, the most important prognostic risk factor for patients with curatively resected primary CRCs.
  • Many tumor-related genes that are epigenetically inactivated in CRC tumor cells by promoter region hypermethylation, such as p16, APC, VHL, and hMLH1 (4-10), have been identified, but the prognostic utility of these methylated genes in primary tumors have not been well described.
  • Our results indicate that ID4 hypermethylation can be used as a prognostic marker for CRC patients.
  • ID4 protein may have a regulatory function. The mechanisms by which the down-regulation of ID4 protein results in unfavorable prognosis are not clear, but it is likely that ID4 down-regulation promotes dedifferentiation and proliferation of CRC cells. ID4 protein may inhibit DNA binding of bHLH transcription factors that are involved in tumor cell dedifferentiation.
  • ID4 of CRC is epigenetically down-regulated. It has been also reported that ID4 is not expressed in certain human breast cancer tissues (41). However, previous animal studies have reported contrary findings showing that ID4 regulates mammary epithelial cell growth and differentiation and is overexpressed in rat mammary gland carcinomas (42). These observations suggest that the regulatory mechanisms of ID4 gene function may be differential in malignant tumors.
  • hypermethylation of the promoter region down-regulates ID4 at the mRNA level in CRC cell lines and at the protein level in clinical specimens.
  • the frequency of hypermethylation is high in primary and metastatic CRCs compared with normal epithelium and adenoma. These results support ID4 as a potential tumor suppressor gene that may play an important role in CRC progression.
  • ID4 transcription inactivation is associated with poorer differentiation of CRC and with unfavorable prognosis.
  • ID4 hypermethylation can be used as a prognostic marker independent of TNM scores or stage of CRC.
  • Vandeputte D A Troost D, Leenstra S, et al. Expression and distribution of id helix-loop-helix proteins in human astrocytic tumors. Glia 2002; 38:329-38.
  • Pagliuca A Cannada-Bartoli P, Lania L. A role for Sp and helix-loop-helix transcription factors in the regulation of the human Id4 gene promoter activity. J Biol Chem 1998; 273:7668-74.
  • ID4 may have a putative tumor suppressive role in early stages of breast cancer progression, and epigenetic inactivation of ID4 gene by promoter hypermethylation might favor regional lymph node metastasis.
  • Tissue specimens and clinicopathology All specimens were from formalin-fixed paraffin-embedded archived tissues (PEAT) of female patients who underwent segmental or total mastectomy with sentinel lymph node biopsy and/or axillary lymph node dissection for T1 invasive breast cancer between 1996 and 2001 at Saint John's Health Center, Santa Monica. Lymph node metastasis was diagnosed with conventional H&E staining. Case-control methodology was adopted to reduce variations associated with tumor size and patient age.
  • PEAT paraffin-embedded archived tissues
  • the N (+) group consisted of 24 patients with axillary lymph node metastasis; the N ( ⁇ ) group consisted of 36 patients without lymph node metastasis, who were selected by matched-sampling to achieve the equivalent distribution of tumor size and age with the N (+) group.
  • the H&E-confirmed normal mammary gland tissues were obtained from the same sections of 11 cancer specimens. Tumors were classified and staged according to the guidelines set by the American Joint Committee on Cancer (AJCC). All patients in this study were consented according to the guidelines set forth by Saint John's Health Center and JWCI human subjects Institutional Review Board (IRB) committee.
  • RNAwiz RNA Isolation Kit (Ambion, Austin, Tex.) following the manufacturer's protocol.
  • the methylation-specific primer set was as follows: forward, 5′-D4-TTTTATAAATATAGTTGCGCGGC-3′; reverse, 5′-GAAACTCCGACTAAACCCGAT-3′.
  • the unmethylation-specific primer set was as follows: forward, 5′-D3-TTTTATAAATATAGTTGTGTGGTGG-3′; reverse, 5′-TCAAAACTCCAACTAAACCCAAT-3′.
  • PCR amplification was performed in a 10-ul reaction volume with 1 ul template for 40 cycles of 30 s at 94° C., 30 s at 58° C., and 30 s at 72° C., followed by a 7-min final extension at 72° C.
  • Mg 2+ concentration was 1.5 mM for methylated-specific and 2.5 mM for unmethylated-specific primer sets.
  • Primer concentration was 0.1 uM for methylated-specific and 0.4 uM for unmethylated-specific primer sets.
  • Reverse-transcriptase reactions were performed on 1.0 ⁇ g of extracted total RNA using Moloney murine leukemia virus reverse-transcriptase (Promega, Madison, Wis.) with oligo-dT primers, as previously described (Takeuchi et al., 2003). For clinical specimens, random primers were additionally used.
  • QRT assay was performed on the iCycler iQ Real-Time thermocycler detection system (Bio-Rad Laboratories, Hercules, Calif.), as previously described (Takeuchi et al., 2003).
  • the reaction mixture consisted of cDNA template synthesized by reverse-transcription from 250 ng of total RNA, 0.5 ⁇ M of forward primer (5′-CGCTCACTGCGCTCAACAC-3′), 0.5 ⁇ M of reverse primer (5′-TCAGGCGGCCGCACACCT-3′), and 0.6 ⁇ M of fluorescence resonance energy transfer (FRET) probe (5′-FAM-CATTCTGTGCCGCTGAGCCG-BHQ-3′).
  • FRET fluorescence resonance energy transfer
  • Absolute copy numbers were determined by a standard curve with serial dilutions (10 8 to 10 0 copies) of DNA containing ID4 cDNA sequence. Analysis without templates was performed as a negative control in each study. PCR products were electrophoresed on 2% agarose gels to confirm product size and absence of non-specific bands. The transcription level of the house-keeping gene GAPDH was measured by qRT as an internal reference with a standard curve to determine the integrity of template RNA for all specimens. The relative mRNA level of ID4 was calculated as: absolute copy number of ID4/absolute copy number of GAPDH in 250 ng of total RNA (Takeuchi et al., 2003). Median values of triplicated quantification were used for analysis.
  • MSP methylation-specific PCR
  • FIG. 6 b Representative MSP results of ID4 promoter region in breast cancer specimens are shown in FIG. 6 b .
  • Hypermethylation of the ID4 promoter region was identified in 16 of 24 (67%) N (+) cancers, 7 of 36 (19%) N ( ⁇ ) cancers, and 2 of 11 (18%) normal mammary gland specimens obtained from the tumor-adjacent tissue ( FIG. 9 ).
  • Tumor size and age were matched because tumor size is one of the most important predictors of tumor behavior in breast cancer (Fisher et al., 1984; Fitzgibbons et al., 2000), and background aberrant hypermethylation increases with age (Toyota & Issa, 1999).
  • Methylation status of cancer specimens was analyzed without knowledge of clinicopathological status, including nodal metastasis. Methylation was objectively determined according to the intensity ratio of methylated-specific and unmethylated-specific peaks quantified by automated capillary array electrophoresis (CAE) system. Because MSP can detect a very small percentage of methylated DNA in abundant unmethylated DNA (Herman et al., 1996), the MSP results would be positive even if only a small part of the microdissected tumor cells was hypermethylated. Frequency of aberrant hypermethylation of N ( ⁇ ) cancer specimens was lower than that of N (+) cancer specimens, which suggested that ID4 gene inactivation might enhanced the aggressiveness and risk of nodal metastasis.
  • CAE capillary array electrophoresis
  • Normal breast specimens showed approximately equivalent base line aberrant hypermethylation level as N ( ⁇ ) cancer specimens. Because the normal tissue was obtained from adjacent normal mammary glands of cancer specimens, there was a possibility that occult cancer cells might be contaminated. There was another possibility that ID4 hypermethylation in pre-malignant mammary gland might be related to the carcinogenesis.
  • ID4 has been shown to be a regulator of BRCA1 expression (Beger et al., 2001), and a negative co-regulation of ID4 and BRCA1 was reported in breast cancer cell lines (Welcsh et al., 2002). However, in primary breast cancers, ID4 expression was observed to be positively correlated with BRCA1 expression (Welcsh et al., 2002). Epigenetic inactivation of ID4 gene may disrupt the ID4-BRCA1 regulatory loop. ID4 was reportedly overexpressed in rat mammary gland carcinomas and positively correlates with proliferation, invasiveness, and tumor weight (Shan et al., 2003). Our results were contradictory to this animal study. Since ID4 can indirectly suppress many kinds of gene expression via downregulating bHLH transcription factors, the mechanism of ID4 function will be complicated. Further functional studies will elucidate the mechanisms of contribution of this gene to breast cancer development and/or progression.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Analytical Chemistry (AREA)
  • Pathology (AREA)
  • Molecular Biology (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Biomedical Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Biophysics (AREA)
  • General Engineering & Computer Science (AREA)
  • Oncology (AREA)
  • Hospice & Palliative Care (AREA)
  • Cell Biology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Toxicology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
US11/345,836 2005-02-01 2006-02-01 Use of ID4 for diagnosis and treatment of cancer Active US7588894B2 (en)

Priority Applications (5)

Application Number Priority Date Filing Date Title
US11/345,836 US7588894B2 (en) 2005-02-01 2006-02-01 Use of ID4 for diagnosis and treatment of cancer
US12/417,577 US7888033B2 (en) 2005-02-01 2009-04-02 Use of ID4 for diagnosis and treatment of cancer
US12/537,509 US20130345144A1 (en) 2005-02-01 2009-08-07 Use of ID4 for Diagnosis and Treatment of Cancer
US12/978,286 US20110098341A1 (en) 2005-02-01 2010-12-23 Use of id4 for diagnosis and treatment of cancer
US14/703,823 US20160122826A1 (en) 2005-02-01 2015-05-04 Use of id4 for diagnosis and treatment of cancer

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US64965005P 2005-02-01 2005-02-01
US11/345,836 US7588894B2 (en) 2005-02-01 2006-02-01 Use of ID4 for diagnosis and treatment of cancer

Related Child Applications (2)

Application Number Title Priority Date Filing Date
US12/417,577 Continuation-In-Part US7888033B2 (en) 2005-02-01 2009-04-02 Use of ID4 for diagnosis and treatment of cancer
US12/537,509 Division US20130345144A1 (en) 2005-02-01 2009-08-07 Use of ID4 for Diagnosis and Treatment of Cancer

Publications (2)

Publication Number Publication Date
US20070020646A1 US20070020646A1 (en) 2007-01-25
US7588894B2 true US7588894B2 (en) 2009-09-15

Family

ID=36777918

Family Applications (3)

Application Number Title Priority Date Filing Date
US11/345,836 Active US7588894B2 (en) 2005-02-01 2006-02-01 Use of ID4 for diagnosis and treatment of cancer
US12/537,509 Abandoned US20130345144A1 (en) 2005-02-01 2009-08-07 Use of ID4 for Diagnosis and Treatment of Cancer
US14/703,823 Abandoned US20160122826A1 (en) 2005-02-01 2015-05-04 Use of id4 for diagnosis and treatment of cancer

Family Applications After (2)

Application Number Title Priority Date Filing Date
US12/537,509 Abandoned US20130345144A1 (en) 2005-02-01 2009-08-07 Use of ID4 for Diagnosis and Treatment of Cancer
US14/703,823 Abandoned US20160122826A1 (en) 2005-02-01 2015-05-04 Use of id4 for diagnosis and treatment of cancer

Country Status (3)

Country Link
US (3) US7588894B2 (fr)
EP (2) EP1869212A4 (fr)
WO (1) WO2006084051A2 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090263812A1 (en) * 2005-02-01 2009-10-22 John Wayne Cancer Institute Use of id4 for diagnosis and treatment of cancer

Families Citing this family (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008077095A2 (fr) * 2006-12-19 2008-06-26 Cornell Research Foundation, Inc. Évaluation de l'état cancéreux par méthylation du gène promoteur de la lécithine-rétinol acyltransférase
US20080221056A1 (en) * 2007-02-12 2008-09-11 Johns Hopkins University Early Detection and Prognosis of Colon Cancers
EP2233590A1 (fr) * 2009-01-28 2010-09-29 AIT Austrian Institute of Technology GmbH Analyse de méthylation
WO2017062863A1 (fr) * 2015-10-09 2017-04-13 Accuragen Holdings Limited Procédés et compositions pour enrichissement de produits d'amplification
IL300974A (en) 2013-12-11 2023-04-01 Accuragen Holdings Ltd Preparations and methods for the detection of rare sequence variants
US11859246B2 (en) 2013-12-11 2024-01-02 Accuragen Holdings Limited Methods and compositions for enrichment of amplification products
US9795660B2 (en) * 2014-12-24 2017-10-24 Children's National Medical Center Id-protein targeted tumor cell vaccine
WO2016185406A1 (fr) 2015-05-19 2016-11-24 Nadathur Estates Pvt. Ltd. Procédé d'identification d'une fonction brca1 déficiente
EP3383994A4 (fr) 2015-12-03 2019-08-28 Accuragen Holdings Limited Méthodes et compositions pour former des produits de ligature
WO2017201102A1 (fr) 2016-05-16 2017-11-23 Accuragen Holdings Limited Procédé de séquençage amélioré par identification de brin
JP6966052B2 (ja) 2016-08-15 2021-11-10 アキュラーゲン ホールディングス リミテッド 稀な配列変異体を検出するための組成物および方法
US10529453B2 (en) * 2017-07-31 2020-01-07 Definiens Gmbh Tool that analyzes image data and generates and displays a confidence indicator along with a cancer score
WO2019076949A1 (fr) * 2017-10-18 2019-04-25 Deutsches Krebsforschungszentrum Moyen et procédés de classification des cancers colorectaux
US11203782B2 (en) 2018-03-29 2021-12-21 Accuragen Holdings Limited Compositions and methods comprising asymmetric barcoding
US12049665B2 (en) 2018-06-12 2024-07-30 Accuragen Holdings Limited Methods and compositions for forming ligation products
CN109659037A (zh) * 2018-12-29 2019-04-19 大连君煜信息技术有限公司 养生方案的推荐方法和装置
CN117821587A (zh) * 2023-12-12 2024-04-05 济南市中心医院 甲基化的id4基因在制备用于诊断幽门螺旋杆菌阳性胃癌产品中的应用

Family Cites Families (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4522811A (en) 1982-07-08 1985-06-11 Syntex (U.S.A.) Inc. Serial injection of muramyldipeptides and liposomes enhances the anti-infective activity of muramyldipeptides
US6017704A (en) 1996-06-03 2000-01-25 The Johns Hopkins University School Of Medicine Method of detection of methylated nucleic acid using agents which modify unmethylated cytosine and distinguishing modified methylated and non-methylated nucleic acids
US6180344B1 (en) * 1997-11-18 2001-01-30 Bin Chen 5′ Upstream region sequences of the MyoD1 gene and uses thereof
US6127178A (en) * 1998-03-20 2000-10-03 The Regents Of The University Of California Apoptotic peptides
US6462187B1 (en) 2000-06-15 2002-10-08 Millennium Pharmaceuticals, Inc. 22109, a novel human thioredoxin family member and uses thereof
US20030040617A9 (en) * 1999-03-12 2003-02-27 Rosen Craig A. Nucleic acids, proteins and antibodies
US6756196B2 (en) 1999-06-28 2004-06-29 Millennium Pharmaceuticals, Inc. Molecules of the card-related protein family and uses thereof
US6649341B1 (en) * 2000-04-19 2003-11-18 Board Of Supervisors Of Louisiana State University And Agricultural And Mechanical College Human glucocorticoid receptor 1A promoter and splice variants
DE60232059D1 (de) * 2001-03-01 2009-06-04 Epigenomics Ag Verfahren zur entwicklung von gensätzen zu diagnostischen und therapeutischen zwecken auf grundlage des expressions- und methylierungsstatus der gene
US7771930B2 (en) * 2001-06-27 2010-08-10 Cancer Research Technology Limited Cancer
US7608696B2 (en) 2001-10-26 2009-10-27 Dana-Farber Cancer Institute, Inc. IBC-1 (invasive breast cancer-1), a putative oncogene amplified in breast cancer
CA2500255A1 (fr) * 2002-10-01 2004-04-29 Epigenomics Ag Procede et acides nucleiques permettant d'ameliorer le traitement de troubles de la proliferation de celllules mammaires

Non-Patent Citations (14)

* Cited by examiner, † Cited by third party
Title
Beger et al., "Identification of Id4 as a Regulator of BRCA1 Expression by Using a Ribozyme-Library-Based Inverse Genomics Approach," Proc. Natl. Acad. Sci. USA, (2001), vol. 98, pp. 130-135.
Chan, et al. Oncogene 2003; 22:6946-6953. *
Das, et al. Journal of Clinical Oncology, 2004; 22(22):4632-4642. *
Fong, et al. Trends in Molecular Medicine 2004; 10(8): 387-392. *
Pagliuca et al., "Molecular Cloning of ID4, a Novel Dominant Negative Helix-Loop-Helix Human Gene on Chromosome 6p21.3-p22," Genomics, (1995), vol. 27, pp. 200-203.
Perk, et al. Nature Reviews Cancer 2005; 5:603-614. *
Rebhan, M., Chalifa-Caspi, V., Prilusky, J., Lancet, D.: GeneCards: encyclopedia for genes, proteins and diseases. Weizmann Institute of Science, Bioinformatics Unit and Genome Center (Rehovot, Israel), 1997. GeneCard for [ID4] [Mar. 22, 2007]). *
Riechmann et al., "Mutually Exclusive Expression of Two Dominant-Negative Helix-Loop-Helix (dnHLH) Genes, Id4 and Id3, in the Developing Brain of the Mouse Suggests Distinct Regulatory Roles of these dnHLH Proteins During Cellular Proliferation and Differentiation of the Nervous System," Cell Growth & Differentiation, (1995), vol. 6, pp. 837-843.
Rigolet et al. cDNA cloning, tissue distribution and chromosomal localization of the human ID4 gene. DNA Research 5(5) : 309-313 (1998). *
Shan et al., "Id4 Regulates Mammary Epithelial Cell Growth and Differentiation and is Overexpressed in Rat Mammary Gland Carcinomas," Am. J. Pathol., (2003), vol. 163, pp. 2495-2502.
Umetani et al., "Allelic Imbalance of APAF-1 Locus at 12q23 is Related to Progression of Colorectal Carcinoma," Oncogene, (2004), vol. 23, pp. 8292-8300.
Umetani, et al. Clin Cancer Res 2004; 10:7475-7483. *
Umetani, et al. Oncogene 2005; 24:4721-4727. *
Welcsh et al., "BRCA1 Transcriptionally Regulates Genes Involved in Breast Tumorigenesis," Proc. Natl. Acad. Sci. USA, (2002), vol. 99, pp. 7560-7565.

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090263812A1 (en) * 2005-02-01 2009-10-22 John Wayne Cancer Institute Use of id4 for diagnosis and treatment of cancer
US7888033B2 (en) * 2005-02-01 2011-02-15 John Wayne Cancer Institute Use of ID4 for diagnosis and treatment of cancer

Also Published As

Publication number Publication date
US20160122826A1 (en) 2016-05-05
EP1869212A2 (fr) 2007-12-26
EP1869212A4 (fr) 2009-09-23
WO2006084051A3 (fr) 2008-10-02
US20130345144A1 (en) 2013-12-26
WO2006084051A2 (fr) 2006-08-10
EP2272984B1 (fr) 2012-06-20
EP2272984A1 (fr) 2011-01-12
US20070020646A1 (en) 2007-01-25

Similar Documents

Publication Publication Date Title
US20160122826A1 (en) Use of id4 for diagnosis and treatment of cancer
Umetani et al. Epigenetic inactivation of ID4 in colorectal carcinomas correlates with poor differentiation and unfavorable prognosis
JP6141896B2 (ja) 選択された遺伝子のエピジェネティックな変化及び癌
Etoh et al. Increased DNA methyltransferase 1 (DNMT1) protein expression correlates significantly with poorer tumor differentiation and frequent DNA hypermethylation of multiple CpG islands in gastric cancers
Gao et al. Genome‐wide promoter methylation analysis identifies epigenetic silencing of MAPK 13 in primary cutaneous melanoma
Pavlidou et al. Diagnostic significance and prognostic role of the ARID1A gene in cancer outcomes
BR112014000443B1 (pt) Métodos para determinação do prognóstico de um indivíduo com câncer
WO2007140319A1 (fr) Promoteurs méthylés utilisés en tant que biomarqueurs du cancer du côlon
BRPI0607508B1 (pt) método para detecção e/ou classificação de distúrbios proliferativos celulares colorretais ou hepatocelulares, e uso do mesmo
EP1989329A2 (fr) Detection et diagnostic de cancers lies au tabagisme
Sharma et al. Prognostic relevance of promoter hypermethylation of multiple genes in breast cancer patients
Patel et al. Characterization of HOXB13 expression patterns in localized and metastatic castration‐resistant prostate cancer
US20120148561A1 (en) Use of runx3 and mir-532-5p as cancer markers and therapeutic targets
Fassan et al. miR-224 is significantly upregulated and targets caspase-3 and caspase-7 during colorectal carcinogenesis
WO2008066878A9 (fr) Procédés et produits utilisés pour diagnostiquer le cancer
Yu et al. Identification of novel subregions of LOH in gastric cancer and analysis of the HIC1 and TOB1 tumor suppressor genes in these subregions
Fu et al. Hypermethylation of APC promoter 1A is associated with moderate activation of Wnt signalling pathway in a subset of colorectal serrated adenomas
Zhong et al. Homozygous deletion of SMAD4 in breast cancer cell lines and invasive ductal carcinomas
US20100143899A1 (en) Methods and products for diagnosing cancer
US7888033B2 (en) Use of ID4 for diagnosis and treatment of cancer
Cai et al. Expression of MET and SOX2 genes in non-small cell lung carcinoma with EGFR mutation
US20110053149A1 (en) Methylation detection in the genomic region of a receptor proteintyrosine phosphatase gamma gene for detection and/or diagnosis of a tumour
Furge et al. Gene expression profiling in kidney cancer: combining differential expression and chromosomal and pathway analyses
Gao Promoter CpG island hypermethylation in the development of cutaneous
Pasculli et al. Predictive Value of Epigenetic Signatures

Legal Events

Date Code Title Description
AS Assignment

Owner name: JOHN WAYNE CANCER INSTITUTE, CALIFORNIA

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:HOON, DAVID S.B.;UMETANI, NAOYUKI;REEL/FRAME:017981/0136;SIGNING DATES FROM 20060607 TO 20060608

STCF Information on status: patent grant

Free format text: PATENTED CASE

FEPP Fee payment procedure

Free format text: PAT HOLDER NO LONGER CLAIMS SMALL ENTITY STATUS, ENTITY STATUS SET TO UNDISCOUNTED (ORIGINAL EVENT CODE: STOL); ENTITY STATUS OF PATENT OWNER: SMALL ENTITY

FEPP Fee payment procedure

Free format text: PAT HOLDER CLAIMS SMALL ENTITY STATUS, ENTITY STATUS SET TO SMALL (ORIGINAL EVENT CODE: LTOS); ENTITY STATUS OF PATENT OWNER: SMALL ENTITY

REMI Maintenance fee reminder mailed
FPAY Fee payment

Year of fee payment: 4

SULP Surcharge for late payment
FPAY Fee payment

Year of fee payment: 8

AS Assignment

Owner name: NATIONAL INSTITUTES OF HEALTH (NIH), U.S. DEPT. OF

Free format text: CONFIRMATORY LICENSE;ASSIGNOR:JOHN WAYNE CANCER INSTITUTE;REEL/FRAME:042303/0750

Effective date: 20170420

AS Assignment

Owner name: NATIONAL INSTITUTES OF HEALTH (NIH), U.S. DEPT. OF

Free format text: CONFIRMATORY LICENSE;ASSIGNOR:JOHN WAYNE CANCER INSTITUTE;REEL/FRAME:042941/0505

Effective date: 20170621

CC Certificate of correction
MAFP Maintenance fee payment

Free format text: PAYMENT OF MAINTENANCE FEE, 12TH YR, SMALL ENTITY (ORIGINAL EVENT CODE: M2553); ENTITY STATUS OF PATENT OWNER: SMALL ENTITY

Year of fee payment: 12